GPCR - (Apr/23/2009 )

hello everyone

I need some suggestion on how to check activity of GPCR when a ligand bind to it, some one suggest me to use GFP gene reporter but I didn't quite understood how it will help
do if you get any ideas please help

thanks

what is GPCR, and what are the downstream effect of ligand binding to GPCR?

G-Protein Coupled Receptor

Will radioactive isotope or FISH work for your purpose?

Search Google Scholar for "GFP GPCR": ~7000 articles, for example: "Using green fluorescent proteins to study
G-protein-coupled receptor localization and trafficking"... Please feel free to read them all.
Cheers,
Minna

fadlone on Apr 23 2009, 10:00 PM said:

A GFP reporter would consist of the GFP gene downstream of a GPCR-responsive element such as cAMP (CRE responsive element), AP1 or NFAT response elements so that when your receptor is activated GFP is expressed and can be observed by fluoresence microscopy or FACS. I found a good review that will help you:


P

Penguin on May 8 2009, 07:03 PM said:

Hi,

If you want to check the activity of GPCR, Simply you can check your recombinant cells in functional assay or in binding assay. In function assay you can do either intracellular ca assay or cAMP assay depending on the predominant signaling pathway. In binding assay you can use radiolabelled ligand to check the receptor.

fadlone on Apr 23 2009, 01:00 PM said:

GFP is a fluorescent visible protein.
GFP coexpresed with GPCR would show you amount of GPRC synthetised.
A GPCR when activated leads to a signal cascade that will lead to the transcription of "fast-response proteins". Those "fast-response proteins" are usually well-known. If you co-express GFP with that "fast-response protein" you would be seeing the cascade effect, extrapolating, the GPCR activity.

If the cell depolarizes upon signal, it means intracelular ion content changes. This changes can be visualize by some fluorescent agents that only fluoresce intracellulary corresponding to the ion content (for ex. FURA2).
This approach is very simple. You mix the cells or tissue with the fluorescent, give the stimuli to the cells and measure fluorescent signal. More and faster fluorescent=more activity.

To probe that protein is the main responsible you can targetedly "deplete-alter" the protein.
Incubate your cells with an antibody against the protein, then with a secondary antibody bound to HRP, finally with tyramide and a bit of H2O2. Several tyramide molecules will bind covalently around were the HRP is, in this case your GPCR, alterating and finishing-reducing it's activity.

Late for the response, but, GPCR's has 3 different routes Gs, Gi and Gq, so the review tells you about how to mesure the signal transduction at any of the steps for the signalling cascade. So, the method most common used is to mesure second messengers, becouse the cell response can be differential, for any 7TM the elicit response, depends on which ligand stabilizes an active conformation, assosiated for the effector domain that interacction with Gs, Gi and Gq, and it could be some cross-talk, like transactivation by RTK's or desensitation by arrestins ie.

So the best you can do, if you do not known which respondes elicits your ligand, is to measure Calcium and cAMP, and add inhibitors for PKA y PKC, to disect the response, and mesure the phosphorilation state of a potential protein targeted by PKA or PKC, or the last protein in the signalling cascade.